Suvrajit Sengupta

The βγ-crystallin superfamily contains both β- and γ-crystallins of the vertebrate eye lens and the microbial calcium-binding proteins, all of which are characterized by a common double-Greek key domain structure. The vertebrate βγ-crystallins are long-lived structural proteins that refract light onto the retina. In contrast, the microbial βγ-crystallins bind calcium ions. The βγ-crystallin from the tunicate Ciona intestinalis (Ci-βγ) provides a potential link between these two functions. It binds calcium with high affinity and is found in a light-sensitive sensory organ that is highly enriched in metal ions. Thus, Ci-βγ is valuable for investigating the evolution of the βγ-crystallin fold away from calcium binding and toward stability in the apo form as part of the vertebrate lens. Here, we investigate the effect of Ca²⁺ and other divalent cations on the stability and aggregation propensity of Ci-βγ and human γS-crystallin (HγS). Beyond Ca²⁺, Ci-βγ is capable of coordinating Mg²⁺, Sr²⁺, Co²⁺, Mn²⁺, Ni²⁺, and Zn²⁺, although only Sr²⁺ is bound with comparable affinity to its preferred metal ion. The extent to which the tested divalent cations stabilize Ci-βγ structure correlates strongly with ionic radius. In contrast, none of the tested divalent cations improved the stability of HγS, and some of them induced aggregation. Zn²⁺, Ni²⁺, and Co²⁺ induce aggregation by interacting with cysteine residues, whereas Cu²⁺-mediated aggregation proceeds via a different binding site.

We report the formation kinetics of trifluoromethane clathrate hydrate (CH) from less than 75 μm diameter ice particles and CHF₃ gas. As previously observed for difluoromethane and propane hydrate formation, the initial stages of the reaction exhibit a strong negative correlation of the reaction rate with temperature, consistent with a negative activation energy of formation. The values obtained for trifluoromethane, ca. −6 kJ/mol (H₂O), are similar to those for difluoromethane, even though the two molecules have different intermolecular interactions and sizes. The activation energy is lesser per mole of H₂O, but greater per mole of guest molecule, than for propane hydrate, which has a different crystal structure. We propose a possible explanation for the negative activation barrier based on the stabilization of metastable structures at low temperature. A pronounced dependence of the formation kinetics on the gas flow rate into the cell is observed. At 253 K and a flow rate of 15 mmol/h, the stage II enclathration of trifluoromethane proceeds so quickly that the overpressure, the difference between the gas cell pressure and the hydrate vapor pressure, is only 0.06 MPa.

The tunicate (Ciona intestinalis) βγ-crystallin represents an intermediate case between the calcium-binding proteins ancestral to the vertebrate βγ-crystallin fold and the vertebrate structural crystallins. Unlike the structural βγ-crystallins in the vertebrate eye lens, this βγ-crystallin strongly binds Ca²⁺. Furthermore, Ca²⁺ binding greatly stabilizes the protein, an effect that has previously been observed in microbial βγ-crystallins but not in those of vertebrates. This relationship between binding and protein stabilization makes the tunicate βγ-crystallin an interesting model for studying the evolution of the human βγ-crystallin. We also compare and contrast the binding sites of tunicate βγ-crystallin with those of other βγ-crystallins to develop hypotheses about the functional origin of the lack of Ca²⁺-binding sites in human crystallins.

The role of methanol as both an inhibitor and a catalyst for the formation of clathrate hydrates (CHs) has been a topic of intense study. We report a new quantitative study of the kinetics of propane CH formation at 253 K from the reaction of propane gas with <75 μm ice particles that have been doped with varying amounts of methanol. We find that methanol significantly accelerates the formation reaction with quite small doping quantities. Even for only 1 methanol molecule per 10 000 water molecules, the maximum uptake rate of propane into CHs is enhanced and the initiation pressure is reduced. These results enable more efficient production of CHs for gas storage. This remarkable acceleration of the CH formation reaction by small quantities of methanol may place constraints on the mechanism of the inhibition effect observed under other conditions, usually employing much larger quantities of methanol.

Many nucleic acids and proteins require divalent metal ions such as Mg²⁺ and Ca²⁺ for folding and function. The lipophilic alignment media frequently used as membrane mimetics also bind these divalent metals. Here we demonstrate the use of ³¹P NMR spectrum of a metal ion chelator (deoxycytidine diphosphate) to measure the bound [Mg²⁺] and [Ca²⁺] in situ for several biological model systems at relatively high divalent ion concentrations (1–10 mM). This method represents a general approach to measuring divalent metal ion binding in NMR samples where the amount and type of metal ion added to the system is known.

Interactions between guest molecules and the water cages in clathrates are dominated by isotropic van der Waals forces at low temperatures because the cage structures satisfy the hydrogen bonding propensity of the water. However, above 200 K the water molecules become more labile and may interact strongly with the guests through hydrogen bonding. In this work we compare the dynamics of tetrahydrofuran (THF) and cyclopentane (CP) guests in the hydrate cages above 200 K. Since THF can form hydrogen bonds while CP cannot, the dynamics provide insight into host–guest hydrogen bonding. We use magic angle spinning (MAS) solid-state NMR to measure proton spin–lattice relaxation times (T₁) of the guests as a function of temperature and find that the activation barrier to the motion of THF molecules is 4.7 kcal/mol (19.7 kJ/mol) at temperatures above 200 K. This is almost 5 times higher than the barrier at lower temperatures. In contrast, the barrier to guest motion in CP hydrate is found to be about 0.67 kcal/mol (2.8 kJ/mol), which agrees well with data at lower temperatures. These results demonstrate that hydrogen bonding interactions between the THF guest and the clathrate cage are significant above 200 K due to the host lattice mobility.

Infectious mammalian prions can be formed de novo from purified recombinant prion protein (PrP) substrate through a pathway that requires the sequential addition of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and RNA cofactor molecules. Recent studies show that the initial interaction between PrP and POPG causes widespread and persistent conformational changes to form an insoluble intermediate species, termed PrP^Int1. Here, we characterize the mechanism and functional consequences of the interaction between POPG and PrP. Negative-stain electron microscopy of PrP^Int1 revealed the presence of amorphous aggregates. Pull-down and photoaffinity label experiments indicate that POPG induces the formation of a PrP^C polybasic-domain-binding neoepitope within PrP^Int1. The ongoing presence of POPG is not required to maintain PrP^Int1 structure, as indicated by the absence of stoichiometric levels of POPG in solid-state NMR measurements of PrP^Int1. Together, these results show that a transient interaction with POPG cofactor unmasks a PrP^C binding site, leading to PrP^Int1 aggregation.